Abstract
The respiratory tract and lungs are subject to diverse pathologies with wide-ranging implications for both human and animal welfare. The development and detailed characterization of cell culture models for studying such forms of disease is of critical importance. In recent years the use of air-liquid interface (ALI)-cultured airway epithelial cells has increased markedly, as this method of culture results in the formation of a highly representative, organotypic in vitro model system. In this study we have expanded on previous knowledge of differentiated ovine tracheal epithelial cells by analysing the progression of differentiation over an extensive time course at an ALI. We observed a pseudo-stratified epithelium with ciliation and a concurrent increase in cell layer thickness from 9 days post-ALI with ciliation approaching a maximum level at day 24. A similar pattern was observed with respect to mucus production with intensely stained PAS-positive cells appearing at day 12. Ultrastructural analysis by SEM confirmed the presence of both ciliated cells and mucus globules on the epithelial surface within this time-frame. Trans-epithelial electrical resistance (TEER) peaked at 1049 Ω × cm2 as the cell layer became confluent, followed by a subsequent reduction as differentiation proceeded and stabilization at ~200 Ω × cm2. Importantly, little deterioration or de-differentiation was observed over the 45 day time-course indicating that the model is suitable for long-term experiments.
Highlights
The primary role of the respiratory system is to conduct air through the nasopharynx, via the trachea, bronchi and bronchioles, into the alveoli for gaseous exchange
General morphological observations and characterization of a variety of cell types including ciliated and goblet cells could be attained by standard hematoxylin and eosin (H&E) staining (Fig 1A), while Periodic Acid Schiff (PAS) staining (Fig 1B) and p63 immunohistochemistry (Fig 1C) allowed for specific labelling of the mucus-producing and basal cell subpopulations, respectively
While the air-liquid interface (ALI) cultures were considerably thinner than the ex vivo tracheal epithelium, the pseudo-stratified morphology associated with these tissues was maintained–the tissue layer was two cells thick from day 12 post-ALI (S4A Fig) and the vast majority of cells maintained contact with the underlying membrane (Fig 1A, 1B and 1C)
Summary
The primary role of the respiratory system is to conduct air through the nasopharynx, via the trachea, bronchi and bronchioles, into the alveoli for gaseous exchange. During the process of inhalation the respiratory system is exposed to a variety of particulates including bacteria, viruses, and pollutants [1,2,3]. The airway epithelium lines the luminal surface of the nasopharynx, trachea, bronchi and bronchioles. It represents the primary point of interaction between inhaled foreign organisms and the host and as such the epithelium has evolved diverse defense mechanisms in order to maintain a virtually sterile environment in the small conducting airways [2, 3]. Successful clearance of particulates depends on an intact, fully functioning epithelial barrier with a complex cellular organization, whereby diverse cell types co-operate in order
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